User:SamBerry/sandbox

Digital Microfluidic Immunoassays

- definition of an immunoassay in the context of digital microfluidics (how it works, sample prep, readouts, advantages/disadvantages)

- applications of digital microfluidic immunoassays and examples for each

Dr. Aaron Wheeler - U Toronto

Add in other researchers doing work in this field

Critique/Edit of "Antibodies" Wikipedia page (Critique an Article)

A few comments and clarifications:

1. A citation for the last sentence in paragraph 3 in the overview is needed, in which the article states, "The allergen-IgE-FcRε interaction mediates allergic signal transduction to induce conditions such as asthma." Induction of asthma is a complex and still not completely understood process, so I think a citation that demonstrates the connection between the antibody interaction and the pathways that lead to asthma or allergic response would be very valuable here. If the citation could also address the previous two sentences as well, that would be even better. I would suggest Williams et al. J Allergy Clin Immunol. 2000 May;105(5):847-59. DOI: 10.1067/mai.2000.106485.

2. Under the Subsection Isotypes, in the second paragraph, the article states, "Immature B cells, which have never been exposed to an antigen, express only the IgM isotype in a cell surface bound form. The B lymphocyte, in this ready-to-respond form, is known as a "naive B lymphocyte"." As it reads, it seems to imply that naive B lymphocytes are also immature B cells, which is false. While immature B cells and naive B lymphocytes have not been exposed to antigens, naive B lymphocytes are mature B lymphocytes, as they have undergone positive selection. I would recommend rewording this so that it isn't as ambiguous and difficult to understand.

SamBerry (talk) 15:56, 6 April 2017 (UTC)

Moved to Sandbox 4/10/17

Addition of Citation to "Antibodies" Wikipedia page (Add to an article)

Added in citation #9as stated above in the critique section.

New Article Addition/Citation - Digital Microfluidics Page (Add to an article)

This citation/phrasing has not yet been added as the page is missing the framework to fit this into it, but it will hopefully be incorporated into the page by the end of the quarter.

[Previous sentence discusses examples of DMF immunoassays that utilize standard colorimetric/fluorescent detection] To further expand the abilities and applications of DMF immunassays beyond colorimetric detection, these devices have been enhanced to utilize alternative methods of detection, such as electrochemical detection using microelectrodes [Following section discusses these papers more in depth (1-2 sentences each about novelty, advantages/disadvantages, etc.)]

Article Draft Section (below)

All initial article drafting was done on a separate Google Doc (class document on Digital Microfluidics) and has since been updated such that the work there is similar/the same as that shown below.

My Article Before Peer Review

The advanced fluid handling capabilities of digital microfluidics (DMF) to precisely manipulate small quantities of liquid reagents allows for the adoption of DMF as an immunoassay (link to immunoassay) platform. Both heterogeneous immunoassays, in which antigens interact with immobilized antibodies, and homogeneous immunoassays, in which antigens interact with antibodies in solution, have been developed using a DMF platform.¹ With regards to heterogeneous immunoassays, DMF can simplify the extended and intensive procedural steps by performing all delivery, mixing, incubation, and washing steps on-chip. Further, existing immunoassay techniques and methods, such as magnetic bead-based assays, ELISAs (link ELISA), and electrochemical detection have been incorporated onto DMF immunoassay platforms.^2,3,4,5 

The incorporation of magnetic bead-based assays onto a DMF immunoassay platform has been demonstrated for the detection of multiple analytes, such as human insulin, IL-6, cardiac marker Troponin I (cTnI), TSH, sTNF-RI, and 17β-estradiol (link all these analytes).^4,6,7,8 For example, Sista et al.⁶ utilized a magnetic bead-based approach for the detection of cTnI from whole blood in less than 8 minutes. Briefly, magnetic beads containing primary antibodies were mixed with labeled secondary antibodies, incubated, and immobilized with a magnet for the washing steps. The droplet was then mixed with a chemiluminescent reagent and detection of the accompanying enzymatic reaction was measured on-chip with a photomultiplier tube.

The ELISA platform is a commonly used technology for performing immunoassays and other biochemical assays that rely on enzyme immunoassays. The ELISA template has been adapted for use with the DMF platform for the detection of analytes such as IgE and IgG.^9,10 Vergauwe et al.² conducted a series of bioassays to establish the quantification capabilities of DMF devices, including an ELISA-based immunoassay for the detection of IgE. Briefly, they used superparamagnetic nanoparticles with immobilized anti-IgE antibodies and fluorescently labeled aptamers to quantify IgE using an ELISA template. Similarly, Zhu et al.⁹ immobilized IgG onto their DMF chip, added horseradish-peroxidase (HRP)-labeled IgG, and then measured the color change associated with product formation of the reaction between HRP and tetramethylbenzidine.

To further expand the capabilities and applications of DMF immunoassays beyond colorimetric detection (i.e., ELISA, magnetic bead-based assays), electrochemical detection tools (e.g., microelectrodes) have been incorporated into DMF chips for the detection of analytes such as thyroid stimulating hormone and rubella virus^5,11,12. For example, Rackus et al.¹¹ integrated microelectrodes onto a DMF chip surface and replaced a previously reported chemiluminescent IgG immunoassay¹³ with an electroactive species, enabling detection of rubella virus. Briefly, they coated magnetic beads with rubella virus, anti-rubella IgG, and anti-human IgG coupled with alkaline phosphatase, which in turn catalyzed an electron transfer reaction that was detected by the on-chip microelectrodes. These methods, in addition to the methods mentioned above, demonstrate the potential of incorporating existing immunoassay technology onto DMF platforms.

References:

1.     Ng A.H.C., Uddayasankar U., Wheeler A.R. “Immunoassays in microfluidic systems”. Anal Bioanal Chem (2010) 39:991-1007. DOI: 10.1007/s00216-010-3678-8.

2.     Vergauwe N., Witters D., Ceyssens F., Vermeir S., Verbruggen B., Puers R., Lammertyn J. “A versatile electrowetting-based digital microfluidic platform for quantitative homogeneous and heterogeneous bio-assays. J. Micromech. Microeng. 21 (2011) 054026. DOI: 10.1088/0960-1317/21/5/054026.

3.     Sista R., Hua Z., Thwar P., Sudarsan A., Srinivasan V., Eckhardt A., Pollack M., Pamula V. “Development of a digital microfluidic platform for point of care testing”. Lab Chip, 2008, 8, 2091-2104. DOI: 10.1039/b814922d.

4.     Ng A.H.C., Choi K., Luoma R.P., Robinson J.M., Wheeler A.R. “Digital Microfluidic Magnetic Separation for Particle-Based Immunoassays”. Anal Chem. 2012, 84, 8805-8812. Dx.doi.org/10.1021/ac3020627.

5.     Shamsi M.H., Choi K., Ng A.H.C., Wheeler A.R. “A digital microfluidic electrochemical immunoassay”. Lab Chip. 2014, 14, 547. DOI: 10.1039/c3lc51063h.

6.     Sista R.S., Eckhardt A.E., Srinivasan V., Pollack M.G., Palanki S., Pamula V.K. “Heterogeneous immunoassays using magnetic beads on a digital microfluidic platform.” Lab Chip. 2008, 8, 2188-2196. DOI: 10.1039/b807855f.

7.     Tsaloglou M.N., Jacobs A., Morgan H. “A fluorogenic heterogenous immunoassay for cardiac muscle troponin cTnI on a digital microfluidic device.” Anal Bioanal Chem. 2014, 406:5967-5976. DOI: 10.1007/s00216-014-7997-z.

8.     Huang C.Y., Tsai P.Y., Lee I.C., Hsu H.Y., Huang H.Y., Fan S.K., Yao D.J., Liu C.H., Hsu W. “A highly efficient bead extraction technique with low bead number for digital microfluidic immunoassay” Biomicro. 2016, 10, 011901. DOI: 10.1063/1.4939942.

9.     Zhu L., Feng Y., Ye X., Feng J., Wu Y., Zhou Z. “An ELISA Chip Based on an EWOD Microfluidic Platform”. J. Adhesion Sci. Tech. 26, 2012, 2113-2124. DOI: 10.1163/156856111x600172.

10.  Miller E.M., Ng A.H.C., Uddayasankar U., Wheeler A.R. “A digital microfluidic approach to heterogeneous immunoassays.” Anal Bioanal Chem. 2011, 399:337-345. DOI: 10.1007/s00216-010-4368-2.

11.  Rackus D.G., Dryden M.D.M., Lamanna J., Zaragoza A., Lam B., Kelley S.O., Wheeler A.R. “A digital microfluidic device with integrated nanostructured microelectrodes for electrochemical immunoassays”. Lab Chip. 2015, 15, 3776. DOI: 10.1039/c5lc00660k.

12.  Dixon C., Ng A.H.C., Fobel R., Miltenburg M.B., Wheeler A.R. “An inkjet printed, roll-coated digital microfluidic device for inexpensive, miniaturized diagnostic assays.” Lab Chip. 2016, 16, 4560. DOI: 10.1039/c6lc01064d.

13.  Ng A.H.C., Lee M., Choi K., Fischer A.T., Robinson J.M, Wheeler A.R. “Digital Microfluidic Platform for the Detection of Rubella Infection and Immunity: A Proof of Concept”. Clin Chem. 2015, 61:2, 420-429. DOI: 10.1373/clinchem.2014.232181

My Article After Peer Review

Digital Microfluidic Immunoassays

The advanced fluid handling capabilities of digital microfluidics (DMF) allows for the adoption of DMF as an immunoassay platform (link to immunoassay), as DMF devices can precisely manipulate small quantities of liquid reagents. Both heterogeneous immunoassays (antigens interacting with immobilized antibodies) and homogeneous immunoassays (antigens interacting with antibodies in solution) have been developed using a DMF platform.¹ With regards to heterogeneous immunoassays, DMF can simplify the extended and intensive procedural steps by performing all delivery, mixing, incubation, and washing steps on the surface of the device (on-chip). Further, existing immunoassay techniques and methods, such as magnetic bead-based assays, ELISAs (link ELISA), and electrochemical detection, have been incorporated onto DMF immunoassay platforms.^2,3,4,5  

The incorporation of magnetic bead-based assays onto a DMF immunoassay platform has been demonstrated for the detection of multiple analytes, such as human insulin, IL-6, cardiac marker Troponin I (cTnI), thyroid stimulating hormone (TSH), sTNF-RI, and 17β-estradiol (link all these analytes).^4,6,7,8 For example, Sista et al.⁶ utilized a magnetic bead-based approach for the detection of cTnI from whole blood in less than 8 minutes. Briefly, magnetic beads containing primary antibodies were mixed with labeled secondary antibodies, incubated, and immobilized with a magnet for the washing steps. The droplet was then mixed with a chemiluminescent reagent and detection of the accompanying enzymatic reaction was measured on-chip with a photomultiplier tube.

The ELISA platform is a commonly used technology for performing immunoassays and other enzyme-based biochemical assays. The ELISA template has been adapted for use with the DMF platform for the detection of analytes such as IgE and IgG.^9,10 Vergauwe et al.² conducted a series of bioassays to establish the quantification capabilities of DMF devices, including an ELISA-based immunoassay for the detection of IgE. In their work, they used superparamagnetic nanoparticles with immobilized anti-IgE antibodies and fluorescently labeled aptamers to quantify IgE using an ELISA template. Similarly, Zhu et al.⁹ immobilized IgG onto their DMF chip, added horseradish-peroxidase (HRP)-labeled IgG, and then measured the color change associated with product formation of the reaction between HRP and tetramethylbenzidine.

To further expand the capabilities and applications of DMF immunoassays beyond colorimetric detection (link to colorimetry) (i.e., ELISA, magnetic bead-based assays), electrochemical detection tools (e.g., microelectrodes) have been incorporated into DMF chips for the detection of analytes such as TSH and rubella virus^5,11,12. For example, Rackus et al.¹¹ integrated microelectrodes onto a DMF chip surface and replaced a previously reported chemiluminescent IgG immunoassay¹³ with an electroactive species, enabling detection of rubella virus. In short, they coated magnetic beads with rubella virus, anti-rubella IgG, and anti-human IgG coupled with alkaline phosphatase, which in turn catalyzed an electron transfer reaction that was detected by the on-chip microelectrodes.

References:

1.     Ng A.H.C., Uddayasankar U., Wheeler A.R. “Immunoassays in microfluidic systems”. Anal Bioanal Chem (2010) 39:991-1007. DOI: 10.1007/s00216-010-3678-8.

2.     Vergauwe N., Witters D., Ceyssens F., Vermeir S., Verbruggen B., Puers R., Lammertyn J. “A versatile electrowetting-based digital microfluidic platform for quantitative homogeneous and heterogeneous bio-assays. J. Micromech. Microeng. 21 (2011) 054026. DOI: 10.1088/0960-1317/21/5/054026.

3.     Sista R., Hua Z., Thwar P., Sudarsan A., Srinivasan V., Eckhardt A., Pollack M., Pamula V. “Development of a digital microfluidic platform for point of care testing”. Lab Chip, 2008, 8, 2091-2104. DOI: 10.1039/b814922d.

4.     Ng A.H.C., Choi K., Luoma R.P., Robinson J.M., Wheeler A.R. “Digital Microfluidic Magnetic Separation for Particle-Based Immunoassays”. Anal Chem. 2012, 84, 8805-8812. Dx.doi.org/10.1021/ac3020627.

5.     Shamsi M.H., Choi K., Ng A.H.C., Wheeler A.R. “A digital microfluidic electrochemical immunoassay”. Lab Chip. 2014, 14, 547. DOI: 10.1039/c3lc51063h.

6.     Sista R.S., Eckhardt A.E., Srinivasan V., Pollack M.G., Palanki S., Pamula V.K. “Heterogeneous immunoassays using magnetic beads on a digital microfluidic platform.” Lab Chip. 2008, 8, 2188-2196. DOI: 10.1039/b807855f.

7.     Tsaloglou M.N., Jacobs A., Morgan H. “A fluorogenic heterogenous immunoassay for cardiac muscle troponin cTnI on a digital microfluidic device.” Anal Bioanal Chem. 2014, 406:5967-5976. DOI: 10.1007/s00216-014-7997-z.

8.     Huang C.Y., Tsai P.Y., Lee I.C., Hsu H.Y., Huang H.Y., Fan S.K., Yao D.J., Liu C.H., Hsu W. “A highly efficient bead extraction technique with low bead number for digital microfluidic immunoassay” Biomicro. 2016, 10, 011901. DOI: 10.1063/1.4939942.

9.     Zhu L., Feng Y., Ye X., Feng J., Wu Y., Zhou Z. “An ELISA Chip Based on an EWOD Microfluidic Platform”. J. Adhesion Sci. Tech. 26, 2012, 2113-2124. DOI: 10.1163/156856111x600172.

10.  Miller E.M., Ng A.H.C., Uddayasankar U., Wheeler A.R. “A digital microfluidic approach to heterogeneous immunoassays.” Anal Bioanal Chem. 2011, 399:337-345. DOI: 10.1007/s00216-010-4368-2.

11.  Rackus D.G., Dryden M.D.M., Lamanna J., Zaragoza A., Lam B., Kelley S.O., Wheeler A.R. “A digital microfluidic device with integrated nanostructured microelectrodes for electrochemical immunoassays”. Lab Chip. 2015, 15, 3776. DOI: 10.1039/c5lc00660k.

12.  Dixon C., Ng A.H.C., Fobel R., Miltenburg M.B., Wheeler A.R. “An inkjet printed, roll-coated digital microfluidic device for inexpensive, miniaturized diagnostic assays.” Lab Chip. 2016, 16, 4560. DOI: 10.1039/c6lc01064d.

13.  Ng A.H.C., Lee M., Choi K., Fischer A.T., Robinson J.M, Wheeler A.R. “Digital Microfluidic Platform for the Detection of Rubella Infection and Immunity: A Proof of Concept”. Clin Chem. 2015, 61:2, 420-429. DOI: 10.1373/clinchem.2014.232181

Reflective Essay

1.    For the Wikipedia final assignment, I worked on the Digital Microfluidics Wikipedia page, which was a preexisting page. This page gave introductory material concerning the history, function, working principle, and use of Digital Microfluidics, but did not go into significant detail regarding the application, variations, or working principles.

2.    My contribution to this page was the addition of an applications section, specifically the section on “Digital Microfluidic Immunoassays”, as well as organization of the other additions to this page from the class. I outlined the location and timeline for the other contributions, and reviewed all the assignments in the group.

3.    The suggestions I received from my peer reviewers were mainly focused on the clarity of my writing, grammar, and wording of complex topics. I accounted for their perspectives while editing my page, incorporating their suggestions into my explanations of concepts. All the feedback was from my peers in the class. For example (see before and after peer review posts in my sandbox),

1.    The first sentence was originally awkwardly worded and a bit long, so I shortened it and made it a little less specific so that it would be appropriate for the general public.

2.    In defining heterogeneous and homogenous immunoassays, I changed the brief definitions to parenthetical definitions to shorten the sentence.

3.    In my three application paragraphs, I used identical structure (and wording) in introducing what was done in each paper, so I changed the wording to be less repetitive.

4.    Overall, I feel that this was a valuable assignment in terms of my learning and as an exercise in reproducing scientific information in an easily understood way. I think going through and doing the literature search and reading was useful and introduced some current and interesting topics that I would have otherwise overlooked, but I believe that writing a review of the papers or being a little less specific would have been more useful. This is due to the fact that writing for Wikipedia did not feel like good practice for scientific writing (for journals, articles, etc.) as it had to be rather broad and non-technical to fit into the associated Wikipedia page. On the other hand, it also was a good exercise in  simplifying scientific concepts and scientific communication. But, I think the scope of the page itself was restricting, as I was including information that needed to be explained but didn’t belong on that page (and hence was linked to the another page). Additionally, I think having a required number of citations made it difficult, as some of the material we were discussing was so specific that there was not an abundance of authors or articles to choose from. That said, I think the exposure of these articles on the Wikipedia page will be helpful to the general public and contribute to their learning as well as their search for materials on the subject.

To improve this assignment in the future, I would broaden the assignment for each individual; instead of allowing students to sign up for a specific part of an existing page, I would suggest 1-2 students work on a page in total. As is, I think the pages will be a little disproportionate, as they will go into extreme detail in some aspects while remain broad in others. By having 1-2 students working on it, you can increase the depth gradually and across the entire article, instead of just in specific parts. There are an abundance of microfluidic pages that could use addition (and even creation), and by spreading out the number of students per page, more pages can be impacted by our work. Lastly, I think there is some disconnect between where we do our work, where (and when) we post it, and what aspects of our work is available to the public. I understand it is important to have a transparent process, but I didn’t feel fully comfortable nor qualified to make edits on a page or post my work in public for all to see. I would suggest doing all the peer reviews, drafts, etc. on a closed Google Doc (between individual and TA/professor) or individual Word documents. I think the privacy would help, and when it was ready to be moved to the main page, we would first post it in our sandbox so that it was available for transparency sake.

The peer reviews were helpful, but they were again an awkward in-between of an academic journal article and the Wikipedia level of information. The mock peer review of an article was straightforward and helpful, but reviewing each other’s articles was more difficult to navigate because of the different expectations between what is posted on Wikipedia and what is published (i.e., I wasn’t sure how critical to be/what standard to hold it against).

Final Draft

Digital Microfluidic Immunoassays

The advanced fluid handling capabilities of digital microfluidics (DMF) allows for the adoption of DMF as an immunoassay platform as DMF devices can precisely manipulate small quantities of liquid reagents. Both heterogeneous immunoassays (antigens interacting with immobilized antibodies) and homogeneous immunoassays (antigens interacting with antibodies in solution) have been developed using a DMF platform. With regards to heterogeneous immunoassays, DMF can simplify the extended and intensive procedural steps by performing all delivery, mixing, incubation, and washing steps on the surface of the device (on-chip). Further, existing immunoassay techniques and methods, such as magnetic bead-based assays, ELISAs, and electrochemical detection, have been incorporated onto DMF immunoassay platforms.

The incorporation of magnetic bead-based assays onto a DMF immunoassay platform has been demonstrated for the detection of multiple analytes, such as human insulin, IL-6, cardiac marker Troponin I (cTnI), thyroid stimulating hormone (TSH), sTNF-RI, and 17β-estradiol. For example,a magnetic bead-based approached has been used for the detection of cTnI from whole blood in less than 8 minutes. Briefly, magnetic beads containing primary antibodies were mixed with labeled secondary antibodies, incubated, and immobilized with a magnet for the washing steps. The droplet was then mixed with a chemiluminescent reagent and detection of the accompanying enzymatic reaction was measured on-chip with a photomultiplier tube.

The ELISA template, commonly used for performing immunoassays and other enzyme-based biochemical assays, has been adapted for use with the DMF platform for the detection of analytes such as IgE and IgG. In one example,a series of bioassays were conducted to establish the quantification capabilities of DMF devices, including an ELISA-based immunoassay for the detection of IgE. Superparamagnetic nanoparticles were  immobilized with anti-IgE antibodies and fluorescently labeled aptamers to quantify IgE using an ELISA template. Similarly, for the detection of IgG, IgG can be immobilized onto a DMF chip, conjugated with horseradish-peroxidase (HRP)-labeled IgG, and then quantified through measurement of the color change associated with product formation of the reaction between HRP and tetramethylbenzidine.

To further expand the capabilities and applications of DMF immunoassays beyond colorimetric detection (i.e., ELISA, magnetic bead-based assays), electrochemical detection tools (e.g., microelectrodes) have been incorporated into DMF chips for the detection of analytes such as TSH and rubella virus. For example, Rackus et al. integrated microelectrodes onto a DMF chip surface and substituted a previously reported chemiluminescent IgG immunoassay with an electroactive species, enabling detection of rubella virus. They coated magnetic beads with rubella virus, anti-rubella IgG, and anti-human IgG coupled with alkaline phosphatase, which in turn catalyzed an electron transfer reaction that was detected by the on-chip microelectrodes.